The emergence of multi-drug-resistant pathogens has become a serious problem in the chemotherapy of bacterial infectious diseases. One of the strategies that can be used to overcome this problem is to find new bacterial protein targets that provide functions essential for cell growth or replication; and to screen for agents that disrupt in some way that essential function.
The process of DNA replication and cell division is potentially an attractive target for antibiotic action. To maintain a consistent number of chromosomes, cells must ensure that during cell division the chromosome is reproducibly replicated, and that each of the new chromosomes is segregated to one of the two daughter cells. Although the ability of bacteria to faithfully replicate and segregate their chromosomes has been appreciated for over forty years, the molecular mechanism by which bacteria execute the segregation process has remained elusive.
Observing the placement and movement of chromosomal loci has revealed that all bacteria examined have highly reproducible, ordered chromosomal structure, with loci exhibiting a linear correlation between their genetic location on the chromosome and their placement along the long axis of the cell. Nonetheless, the details of chromosomal topology vary among species. For example, the rod-shaped bacteria E. coli and B. subtilis localize their origins of replication to the mid-cell and quarter-cell positions at different stages of the cell cycle, whereas in the crescent-shaped Caulobacter crescentus chromosomal origins are at the cell poles. Direct examination of chromosome segregation in live cells revealed that the origin region (defined as the site of the initiation of DNA replication) is the first region of the chromosome to be segregated in all species examined.
The rapid, directed movement of the origins excludes the cell growth-related mechanisms initially proposed for chromosome segregation and is suggestive of an active mechanism for origin transport. Several candidates have been put forth as potential contributors to the force that separates the chromosomes. The coincident timing of DNA replication and segregation suggests that these processes are coupled and that the act of DNA replication could provide the motive force for chromosome segregation. The observation that the DNA replication machinery is stationary in B. subtilis and E. coli led to the refinement of such models into the “extrusion-capture” model, wherein the extrusion of DNA from an immobile replisome forces it poleward, where it is then captured by as-yet unidentified factors. Coordinate transcription of origin-proximal reading frames oriented away from the origin has also been proposed to contribute to the movement of bacterial chromosomes. Though these models are compelling and elegant, they lack experimental validation.
In contrast, the mechanisms directing eukaryotic chromosome segregation are well characterized. Eukaryotes use a microtubule-based cytoskeletal system for chromosome partitioning. After replication, chromosome sisters are paired, and microtubules are attached to specific, centromeric chromosomal loci through a kinetochore protein complex. Once attached, the microtubules direct chromosome migration to opposite poles through dynamic polymerization cycles and the action of motor proteins. Bacterial proteins that could provide a function analogous to the kinetochore protein complex would be an attractive target for antibiotic action.
There is a clinical need for novel antibiotic agents. The present invention addresses this need.
Selected Literature Citations
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